The SSX genes are located on the X chromosome and encode a family of highly homologous nuclear proteins. Two family members, SSX-1 and SSX-2, were initially identified as fusion partners of the SYT gene in t(X;18)-positive synovial sarcomas (Clark, J., et al., 1994, Identification of novel genes, SYT and SSX, involved in the t(X;18)(p11.2;q11.2) translocation found in human synovial sarcoma, Nat Genet, 7:502-508; Crew, A. J., et al., 1995, Fusion of SYT to two genes, SSX1 and SSX2, encoding proteins with homology to the Kruppel-associated box in human synovial sarcoma, Embo J, 14:2333-2340). Later, serological analysis of tumor cDNA expression libraries (SEREX), revealed recognition of the SSX-2 encoded antigen by antibodies from cancer patients (Tureci, O. et al., 1996, The SSX-2 gene, which is involved in the t(X;18) translocation of synovial sarcomas, codes for the human tumor antigen HOM-MEL-40, Cancer Res, 56:4766-4772). Three additional homologous genes, SSX-3, -4 and -5, were identified (de Leeuw, B. et al., 1996, A novel Kruppel-associated box containing the SSX gene (SSX3) on the human X chromosome is not implicated in t(X;18)-positive synovial sarcomas, Cytogenet Cell Genet, 73:179-183; Gure, A. O. et al., 1997, SSX: a multigene family with several members transcribed in normal testis and human cancer, Int J Cancer, 72:965-971). Recently, SSX genes and pseudogenes have been further characterized, resulting in the definition of 9 (SSX-1 to -9) genes (Gure, A. O. et al., 2002, The SSX gene family: Characterization of 9 complete genes, Int J Cancer, 101:448-453). Similarly to other members of the cancer/testis antigens (CTA) group, to which the SSX gene family belongs, expression of the majority of the SSX genes, including SSX-1 to −5 and SSX-7, is restricted to gametogenic cells but silent in most adult normal tissues (Scanlan, M. J. et al., 2002, Cancer/testis antigens: an expanding family of targets for cancer immunotherapy, Immunol Rev, 188:22-32). Importantly, expression of these antigens is also detected in tumors of different histological types (Tureci, O. et al., 1998, Expression of SSX genes in human tumors, Int J Cancer, 77:19-23; Naka, N. et al., 2002, Expression of SSX genes in human osteosarcomas, Int J Cancer, 98:640-642; Chen, C. H., et al., 2001, Expressions of cancer-testis antigens in human hepatocellular carcinomas, Cancer Lett, 164:189-195; Ayyoub, M., et al., 2003, SSX antigens as tumor vaccine targets in human sarcoma, Cancer Immunity, 3:13). Therefore, antigens of the SSX family are targets of great interest for immunotherapy of cancer.
Among SSX genes, SSX-1, -2, -4 and -5 are the most commonly expressed. Several surveys of SSX gene expression in different human tumor types showed expression of several family members in a significant proportion of tumors, although at variable levels depending on the particular histological type. Expression of at least one SSX family member was frequently observed in tumor types such as head and neck cancer (75%), ovarian cancer (50%), malignant melanoma (43%) (Tureci, O. et al., 1998, Expression of SSX genes in human tumors, Int J Cancer, 77:19-23) and sarcoma (42%) (Naka, N. et al., 2002, Expression of SSX genes in human osteosarcomas, Int J Cancer, 98:640-642; Ayyoub, M., et al., 2003, SSX antigens as tumor vaccine targets in human sarcoma, Cancer Immunity, 3:13). We and others have previously reported that one antigen of the family, SSX-2, is naturally immunogenic in cancer patients bearing antigen-expressing tumors, and stimulates both specific humoral and T cell responses. Using tumor-reactive CD8+ T lymphocytes from a melanoma patient we identified a CTL epitope restricted by the frequently expressed MHC class I allele HLA-A2 (Ayyoub, M., et al., 2002, Proteasome-assisted identification of a SSX-2-derived epitope recognized by tumor-reactive CTL infiltrating metastatic melanoma, J Immunol, 168:1717-1722; Rubio-Godoy, V., et al., 2002, Combinatorial peptide library-based identification of peptide ligands for tumor-reactive cytolytic T lymphocytes of unknown specificity, Eur J Immunol, 32:2292-2299). High affinity CD8+ T cell responses to this epitope were specifically found in HLA-A2+ melanoma patients bearing SSX-2 expressing tumors (Ayyoub, M., et al., 2003, Tumor-reactive SSX-2-specific CD8+ T cells are selectively expanded during immune responses to antigen expressing tumors in melanoma patients, Cancer Res, 63:5601-5606). More recently, we have reported the identification of three SSX-2 derived CD4+ T cell epitopes recognized by specific T cells isolated from antigen expressing melanoma patients (Ayyoub, M., et al., 2004, Identification of an SSX-2 epitope presented by dendritic cells to circulating autologous CD4+ T cells, J Immunol, 172:7206-7211; Ayyoub, M., et al., 2004, An immunodominant SSX-2-derived epitope recognized by CD4+ T cells in association with HLA-DR, J Clin Invest, 113:1225-1233). No information, however, was thus far available on the immunogenicity of other SSX antigens.
Here, we report the analysis of naturally occurring CD4+ T cell responses against another frequently expressed SSX antigen, SSX-4, in melanoma patients. Upon in vitro stimulation with a pool of long peptides spanning the protein sequence, we could detect and isolate SSX-4 specific CD4+ T cells from 4/4 melanoma patients bearing antigen-expressing tumors but not from healthy individuals. From circulating CD4+ T lymphocytes from these patients we isolated SSX-4 specific clonal CD4+ T cell populations recognizing 7 distinct epitopes restricted by 5 different HLA class II alleles including some among the HLA-DR alleles more frequently expressed in several major ethnic groups. Interestingly, the majority of the identified epitopes were located within the Krüppel associated box (KRAB) repression domain in the N-terminal region of the protein. Together, the results of our study reveal a high spontaneous immunogenicity of SSX-4 and support the inclusion of this antigen in immunotherapy trials for melanoma and other SSX-expressing cancers.